Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (10): 43-50    DOI: 10.13523/j.cb.2005035
综述     
CRISPR/Cas9系统的发展及其在动物基因编辑中的应用 *
王志敏,毕美玉,贺佳福,任炳旭,刘东军()
内蒙古大学省部共建草原家畜生殖调控与繁育国家重点实验室 呼和浩特 010070
Development of CRISPR/Cas9 System and Its Application in Animal Gene Editing
WANG Zhi-min,BI Mei-yu,HE Jia-fu,Ren Bing-xu,LIU Dong-jun()
State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010070, China
 全文: PDF(936 KB)   HTML
摘要：

CRISPR/Cas9作为一种新型的基因编辑技术,主要在DNA水平对生物体的遗传信息进行修改,具有强大的基因编辑能力,目前已经被广泛应用于多个领域,包括基因功能研究、构建动物模型、家畜新品种的培育以及基因治疗等。CRISPR/Cas9技术的不断发展为生物学及医学领域的研究带来了革命性的突破,利用该技术构建基因突变小鼠有助于基因功能的研究,对于遗传疾病的治疗等具有重要的参考价值,同时还可以从基因组水平上有效改善家畜动物的生产性能,提高家畜动物的抗病能力。主要介绍了CRISPR/Cas系统的研究历程、结构与分类,详细阐述了CRISPR/ Cas9技术的作用机制及其在动物基因编辑中的应用,探讨了CRISPR/Cas9在基因编辑动物的制备中存在的问题及优化策略,并对其发展前景进行了展望。
关键词： CRISPR/Cas9;  基因编辑;  动物;  家畜育种    
Abstract:

CRISPR/Cas9, a new gene editing technology, mainly modifies the genetic information of organisms at the DNA level and has powerful gene editing ability. Now, it has been widely used in many fields, including gene function research, animal model construction, new breed breeding and gene therapy. The continuous development of CRISPR/Cas9 technology has brought a revolutionary breakthrough in the field of biology and medicine. Using this technology to construct gene mutant mice is not only conducive to the research of gene function, but also has an important reference value for the treatment of genetic diseases. In addition, this technology can effectively improve the production performance of livestock at the molecular level, and improve the disease resistance of livestock. Mainly introduces the research process, structure and classification of CRISPR/Cas system, expounds the mechanism of CRISPR/Cas9 technology and its application in animal gene editing, discusses the problems and optimization strategies of CRISPR/Cas9 in making gene editing animals, and prospects the development of CRISPR/Cas9.
Key words: CRISPR/Cas9    Gene editing    Animal    Livestock breeding
收稿日期: 2020-05-18 出版日期: 2020-11-10
ZTFLH:  Q819  
基金资助: * 内蒙古自治区科技创新引导项目(KCBJ2018033);内蒙古自治区科技厅重大专项(ZDZX2018065)
通讯作者: 刘东军     E-mail: nmliudongjun@sina.com
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
王志敏
毕美玉
贺佳福
任炳旭
刘东军

引用本文:

王志敏,毕美玉,贺佳福,任炳旭,刘东军. CRISPR/Cas9系统的发展及其在动物基因编辑中的应用 *[J]. 中国生物工程杂志, 2020, 40(10): 43-50.

WANG Zhi-min,BI Mei-yu,HE Jia-fu,Ren Bing-xu,LIU Dong-jun. Development of CRISPR/Cas9 System and Its Application in Animal Gene Editing. China Biotechnology, 2020, 40(10): 43-50.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2005035    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I10/43

图1  CRISPR/Cas系统基本结构
[1] Sander J D, Dahlborg E J, Goodwin M J, et al. Selection-free zinc-finger nuclease engineering by context-dependent assembly (CoDA). Nature Methods, 2011,8(1):67-69.
doi: 10.1038/nmeth.1542 pmid: 21151135
[2] Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012,337(6096):816-821.
doi: 10.1126/science.1225829 pmid: 22745249
[3] Ishino Y, Shinagawa H, Makino K, et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol, 1987,169(12):5429-5433.
pmid: 3316184
[4] Mojica F J M, Juez G, Rodríguez-Valera F. Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites. Mol Microbiol, 1993,9(3):613-621.
doi: 10.1111/j.1365-2958.1993.tb01721.x pmid: 8412707
[5] Mojica F J M, Diez-Villasenor C, Soria E, et al. Biological significance of a family of regularly spaced repeats in the genomes of archaea, bacteria and mitochondria. Mol Microbiol, 2000,36(1):244-246.
doi: 10.1046/j.1365-2958.2000.01838.x pmid: 10760181
[6] Jansen R Embden J D A V Gaastra W, et al. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol, 2002,43(6):1565-1575.
doi: 10.1046/j.1365-2958.2002.02839.x pmid: 11952905
[7] Mojica F J M, Díez-Villasenor C, García-Martínez J, et al. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol, 2005,60(2):174-182.
doi: 10.1007/s00239-004-0046-3 pmid: 15791728
[8] Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology, 2005,151(3):653-663.
[9] Marraffini L A, Sontheimer E J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science, 2008,322(5909):1843-1845.
doi: 10.1126/science.1165771 pmid: 19095942
[10] Garneau J E, Dupuis M-E, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010,468(7320):67-71.
doi: 10.1038/nature09523 pmid: 21048762
[11] Deltcheva E, Chylinski K, Sharma C M, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 2011,471(7340):602-607.
doi: 10.1038/nature09886 pmid: 21455174
[12] Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A, 2012,109(39):2579-2586.
[13] Le C, Ran F A, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013,339(6121):819-823.
doi: 10.1126/science.1231143 pmid: 23287718
[14] Suzuki K, Tsunekawa Y, Hernandez-Benitez R, et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature, 2016,540(7631):144-149.
doi: 10.1038/nature20565 pmid: 27851729
[15] Frieda K L, Linton J M, Hormoz S, et al. Synthetic recording and in situ readout of lineage information in single cells. Nature, 2017,541(7635):107-111.
doi: 10.1038/nature20777 pmid: 27869821
[16] Deveau H, Garneau J E, Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions. Annual Review of Microbiology, 2010,64(1):475-493.
[17] Shah S A, Erdmann S, Mojica F J, et al. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biology, 2013,10(5):891-899.
doi: 10.4161/rna.23764 pmid: 23403393
[18] Sorek R, Kunin V, Hugenholtz P. CRISPR- a widespread system that provides acquired resistance against phages in bacteria and archaea. Nature Reviews Microbiology, 2008,6(3):181-186.
doi: 10.1038/nrmicro1793 pmid: 18157154
[19] Makarova K S, Aravind L, Grishin N V, et al. A DNA repair system specific for thermophilic archaea and bacteria predicted by genomic context analysis. Nucleic Acids Research, 2002,30(2):482-496.
doi: 10.1093/nar/30.2.482 pmid: 11788711
[20] Jinek M, Jiang F, Taylor D W, et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science, 2014,343(6176):1247997.
doi: 10.1126/science.1247997 pmid: 24505130
[21] Sinkunas T, Gasiunas G, Fremaux C, et al. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. Embo Journal, 2014,30(7):1335-1342.
doi: 10.1038/emboj.2011.41 pmid: 21343909
[22] Wiedenheft B, van Duijn E, Bultema J B, et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proceedings of the National Academy of Sciences of the United States of America, 2011,108(25):10092-10097.
[23] Anantharaman V, Iyer L M, Aravind L. Presence of a classical RRM-fold palm domain in Thg1- type 3'- 5' nucleic acid polymerases and the origin of the GGDEF and CRISPR polymerase domains. Biology Direct, 2010,5(1):43.
[24] Sapranauskas R, Gasiunas G, Fremaux C, et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Research, 2011,39(21):9275-9282.
doi: 10.1093/nar/gkr606 pmid: 21813460
[25] Datsenko K A, Pougach K, Tikhonov A, et al. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nature Communications, 2012,3(1):945.
[26] Heler R, Samai P, Modell J W, et al. Cas9 specifies functional viral targets during CRISPR-Cas adaptation. Nature, 2015,519(7542):199-202.
doi: 10.1038/nature14245 pmid: 25707807
[27] Ran F A, Hsu P D, Wright J, et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 2013,8(2281):308.
[28] Shi J, Zhao Y, Wang K, et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature, 2015,526(7575):660-665.
doi: 10.1038/nature15514 pmid: 26375003
[29] Zuckermann M, Hovestadt V, Knobbe-Thomsen C B, et al. Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling. Nature Communications, 2015,6:7391.
doi: 10.1038/ncomms8391 pmid: 26067104
[30] Platt R J, Chen S, Zhou Y, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell, 2014,159(2):440-455.
doi: 10.1016/j.cell.2014.09.014 pmid: 25263330
[31] Carroll K J, Makarewich C A, Mcanally J, et al. A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9. Proc Natl Acad Sci USA, 2016,113(2):338-343.
doi: 10.1073/pnas.1523918113 pmid: 26719419
[32] Wang X, Raghavan A, Chen T, et al. CRISPR-Cas9 targeting of PCSK9 in human hepatocytes in vivo. Arteriosclerosis Thrombosis Vascular Biology, 2016,36(5):783.
[33] Gao X, Tao Y, Lamas V, et al. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature, 2018,553(7687):217-221.
pmid: 29258297
[34] Yu W, Mookherjee S, Chaitankar V, et al. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nature Communications, 2017,8:14716.
doi: 10.1038/ncomms14716 pmid: 28291770
[35] Dewitt M A, Magis W, Bray N L, et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Science Translational Medicine, 2016,8(360):134.
[36] Nelson C E, Wu Y, Gemberling M P, et al. Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nature Medicine, 2019,25(3):427-432.
doi: 10.1038/s41591-019-0344-3 pmid: 30778238
[37] Wang L, Yang Y, White J, et al. CRISPR/Cas9-mediated in vivo gene targeting corrects haemostasis in newborn and adult FIX-KO mice. Blood, 2016,128(22):1174-1174.
[38] Meng F, Zhao D, Zhou Q, et al. Construction of EZH2 knockout animal model by CRISPR/Cas9 technology. Zhongguo Fei Ai Za Zhi, 2018,21(5):358-364.
doi: 10.3779/j.issn.1009-3419.2018.05.02 pmid: 29764585
[39] Wang K, Ouyang H, Xie Z, et al. Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system. Scientific Reports, 2015,5:16623.
doi: 10.1038/srep16623 pmid: 26564781
[40] Crispo M, Mulet A P, Tesson L, et al. Efficient generation of myostatin knock-out sheep using CRISPR/Cas9 technology and microinjection into zygotes. PLoS One, 2015,10(8):e0136690.
pmid: 26305800
[41] Wang X, Niu Y, Zhou J, et al. CRISPR/Cas9 ediated MSTN disruption and heritable mutagenesis in goats causes increased body mass. Animal Genetics, 2018,49(1):43-51.
doi: 10.1111/age.12626 pmid: 29446146
[42] Liu X, Liu H, Wang M, et al. Disruption of the ZBED6 binding site in intron 3 of IGF2 by CRISPR/Cas9 leads to enhanced muscle development in liang guang small spotted pigs. Transgenic Research, 2019,28(1):141-150.
doi: 10.1007/s11248-018-0107-9 pmid: 30488155
[43] Xiang G, Ren J, Hai T, et al. Editing porcine IGF2 regulatory element improved meat production in Chinese Bama pigs. Cellular Molecular Life Sciences, 2018,75(24):4619-4628.
doi: 10.1007/s00018-018-2917-6 pmid: 30259067
[44] Ni W, Qiao J, Hu S, et al. Efficient gene knockout in goats using CRISPR/Cas9 system. PLoS One, 2014,9(9):e106718.
doi: 10.1371/journal.pone.0106718 pmid: 25188313
[45] Zheng Q, Lin J, Huang J, et al. Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity. Proc Natl Acad Sci U S A, 2017,114(45):e9474-9482.
doi: 10.1073/pnas.1707853114 pmid: 29078316
[46] Zhou Y, Lin Y, Wu X, et al. The high-level accumulation of n-3 polyunsaturated fatty acids in transgenic pigs harboring the n-3 fatty acid desaturase gene from Caenorhabditis briggsae. Transgenic Research, 2014,23(1):89-97.
doi: 10.1007/s11248-013-9752-1 pmid: 24048769
[47] Yu S, Luo J, Song Z, et al. Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle. Cell Research, 2011,21(11):1638-1640.
doi: 10.1038/cr.2011.153 pmid: 21912434
[48] Cui C, Song Y, Liu J, et al. Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk. Scientific Reports, 2015,5:10482.
doi: 10.1038/srep10482 pmid: 25994151
[49] Jeong Y H, Kim Y J, Kim E Y, et al. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination. Zygote, 2015,24(3):442-456.
doi: 10.1017/S0967199415000374 pmid: 26197710
[50] Peng J, Wang Y, Jiang J, et al. Production of human albumin in pigs through CRISPR/Cas9-mediated knockin of human cDNA into swine albumin locus in the zygotes. Scientific Reports, 2015,5(16705):1-5.
[51] Richt J A, Kasinathan P, Hamir A N, et al. Production of cattle lacking prion protein. Nature Biotechnology, 2007,25(1):132-138.
doi: 10.1038/nbt1271 pmid: 17195841
[52] Burkard C, Opriessnig T, Mileham A J, et al. Pigs lacking the scavenger receptor cysteine-rich domain 5 of CD163 are resistant to PRRSV-1 infection. J Virol, 2018,92(16):e00415-00418.
doi: 10.1128/JVI.00415-18 pmid: 29925651
[53] Xie Z, Pang D, Yuan H, et al. Genetically modified pigs are protected from classical swine fever virus. PLoS Pathog, 2018,14(12):e1007193.
doi: 10.1371/journal.ppat.1007193 pmid: 30543715
[54] Lu C, Pang D X, Li M J, et al. CRISPR/Cas9-mediated hitchhike expression of functional shRNAs at the porcine miR-17-92 cluster. Cell, 2019,8(2):113-129.
[55] Xu A, Qin C, Lang Y, et al. A simple and rapid approach to manipulate pseudorabies virus genome by CRISPR/Cas9 system. Biotechnology Letters, 2015,37(6):1265-1272.
doi: 10.1007/s10529-015-1796-2 pmid: 25724716
[56] Tang Y D, Liu J T, Wang T Y, et al. CRISPR/Cas9-mediated multiple single guide RNAs potently abrogate pseudorabies virus replication. Archives of Virology, 2017,162(12):1-6.
[57] Gao Y, Wu H, Wang Y, et al. Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects. Genome Biology, 2017,18(1):13.
doi: 10.1186/s13059-016-1144-4 pmid: 28143571
[58] Fu Y, Sander J D, Reyon D, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nature Biotechnology, 2014,32(3):279-284.
pmid: 24463574
[59] Müller M, Lee C M, Gasiunas G, et al. Streptococcus thermophilus CRISPR-Cas9 systems enable specific editing of the human genome. Molecular Therapy, 2016,24:636-644.
doi: 10.1038/mt.2015.218 pmid: 26658966
[60] Labuhn M, Adams F F, Ng M, et al. Refined sgRNA efficacy prediction improves large- and small-scale CRISPR/Cas9 applications. Nucleic Acids Research, 2018,46(3):1375-1385.
doi: 10.1093/nar/gkx1268 pmid: 29267886
[61] Kim D, Kim S, Kim S, et al. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Research, 2016,26(3):406-415.
doi: 10.1101/gr.199588.115 pmid: 26786045
[62] Slaymaker I, Gao F, Zetsche B, et al. Rationally engineered Cas9 nucleases with improved specificity. Science, 2016,351:84-88.
doi: 10.1126/science.aad5227 pmid: 26628643
[63] Bolukbasi M F, Gupta A, Oikemus S, et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nature Methods, 2015,12:1150-1156.
doi: 10.1038/nmeth.3624 pmid: 26480473
[64] Wyvekens N, Topkar V V, Khayter C, et al. Dimeric CRISPR RNA-guided FokI-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing. Human Gene Therapy, 2015,26(7):425-431.
doi: 10.1089/hum.2015.084 pmid: 26068112
[65] Kim S, Kim D, Cho S W, et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research, 2014,24(6):1012-1019.
doi: 10.1101/gr.171322.113 pmid: 24696461
[66] Hsu P D, Scott D A, Weinstein J A, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology, 2013,31(9):827.
doi: 10.1038/nbt.2647 pmid: 23873081
[67] Shin J, Jiang F, Liu J, et al. Disabling Cas9 by an anti-CRISPR DNA mimic. Science Adv, 2017,3(7):e1701620.
[68] Wang J Z, Wu P, Shi Z M, et al. The AAV-mediated and RNA-guided CRISPR/Cas9 system for gene therapy of DMD and BMD. Brain Development, 2017,39:547-556.
doi: 10.1016/j.braindev.2017.03.024 pmid: 28390761
[69] Timin A S, Muslimov A R, Lepik K V, et al. Efficient gene editing via non-viral delivery of CRISPR-Cas9 system using polymeric and hybrid microcarriers. Nanomedicine Nanotechnology Biology Medicine, 2018,14:97-108.
[70] Liu C, Zhang L, Liu H, et al. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. Journal of Controlled Release, 2017,266(7):17-26.
[71] Dewitt M A, Corn J E, Carroll D. Genome editing via delivery of Cas9 ribonucleoprotein. Methods, 2017, 121-122:9-15.
doi: 10.1016/j.ymeth.2017.04.003 pmid: 28410976
[72] Ma Y, Chen W, Zhang X, et al. Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing in rats by inhibiting NHEJ and using Cas9 protein. RNA Biology, 2016,13:605-612.
doi: 10.1080/15476286.2016.1185591 pmid: 27163284
[73] He X, Tan C, Feng W, et al. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Research, 2016,44:e85.
doi: 10.1093/nar/gkw064 pmid: 26850641
[74] Suzuki K, Tsunekawa Y, Hernandez-Benitez R, et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature, 2016,540(7631):144-149.
doi: 10.1038/nature20565 pmid: 27851729
[75] Yang D, Scavuzzo M A, Chmielowiec J, et al. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Scientific Reports, 2016,6:21264.
doi: 10.1038/srep21264 pmid: 26887909
[76] Hendel A, Bak R O, Clark J T, et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nature Biotechnology, 2015,33(9):985-989.
doi: 10.1038/nbt.3290 pmid: 26121415
[1] 武秀知,王宏杰,祖尧. 斑马鱼hoxa1a基因调控颅面骨骼发育的功能研究*[J]. 中国生物工程杂志, 2021, 41(9): 20-26.
[2] 毕博,张宇,赵慧. 酵母杂交系统在CRISPR/Cas9基因编辑系统脱靶率研究中的应用*[J]. 中国生物工程杂志, 2021, 41(6): 27-37.
[3] 胡暄,王松,于学玲,张晓鹏. 不稳定EGFP细胞模型的构建及其在基因编辑体系评价中的应用*[J]. 中国生物工程杂志, 2021, 41(5): 17-26.
[4] 王艳梅,寇航,马梅,申玉玉,赵宝顶,路福平,黎明. 利用CRISPR-Cas9技术失活黑曲霉中果胶酶基因及突变株性能评价*[J]. 中国生物工程杂志, 2021, 41(5): 35-44.
[5] 王惠临,周凯强,朱红雨,王力景,杨仲璠,徐明波,曹荣月. 凝血因子VII及其重组表达新进展[J]. 中国生物工程杂志, 2021, 41(2/3): 129-137.
[6] 冷燕,孙康泰,刘倩倩,蒲阿庆,李翔,万向元,魏珣. 全球基因编辑作物监管趋势研究[J]. 中国生物工程杂志, 2021, 41(12): 24-29.
[7] 郭洋,陈艳娟,刘怡辰,王海杰,王成稷,王珏,万颖寒,周宇,奚骏,沈如凌. Pd-1基因敲除小鼠构建及初步表型验证[J]. 中国生物工程杂志, 2021, 41(10): 1-11.
[8] 郭洋,万颖寒,王珏,龚慧,周宇,慈磊,万志鹏,孙瑞林,费俭,沈如凌. Toll样受体4(TLR4)基因剔除小鼠构建及初步表型分析[J]. 中国生物工程杂志, 2020, 40(6): 1-9.
[9] 樊斌,陈欢,宋婉莹,陈光,王刚. 乳酸菌基因改造技术研究进展 *[J]. 中国生物工程杂志, 2020, 40(6): 84-92.
[10] 刘迪,张洪春. 慢性阻塞性肺疾病基因工程动物模型研究进展 *[J]. 中国生物工程杂志, 2020, 40(4): 59-68.
[11] 黄胜, 严启滔, 熊仕琳, 彭弈骐, 赵蕊. 基于CRISPR/Cas9-SAM系统CHD5基因过表达慢病毒载体的构建及对膀胱癌T24细胞增殖,迁移和侵袭能力的影响 *[J]. 中国生物工程杂志, 2020, 40(3): 1-8.
[12] 雷海英,赵青松,白凤麟,宋慧芳,王志军. 利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*[J]. 中国生物工程杂志, 2020, 40(12): 49-57.
[13] 王玥,牟彦双,刘忠华. 基于CRISPR/Cas系统的单碱基编辑技术研究进展*[J]. 中国生物工程杂志, 2020, 40(12): 58-66.
[14] 王伟东,杜加茹,张运尚,樊剑鸣. CRISPR/Cas9在人病毒感染相关疾病治疗研究中的应用*[J]. 中国生物工程杂志, 2020, 40(12): 18-24.
[15] 何秀娟,胡凤枝,刘秋丽,刘玉萍,祝玲,郑文云. 乳腺癌细胞QSOX1的CRISPR/Cas9基因编辑及其对增殖侵袭的影响研究*[J]. 中国生物工程杂志, 2020, 40(11): 1-9.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会